Group B Streptococcus (GBS) is a leading cause of infectious morbidity during infancy. Late-onset GBS infection (>7 days of age) typically manifests as sepsis, focal bone/soft tissue infections, or meningitis, all of which may be associated with significant long-term sequelae in survivors. In contrast to early-onset GBS infection, there are currently no effective strategies to reduce the incidence of late-onset GBS disease or to identify those infants who may be most at risk. We have developed a novel murine model of late-onset GBS infection that closely mimics the human disease state in which perinatally acquired GBS first colonizes the newborn intestinal tract and then transverses intestinal barriers to cause invasive disease. In Aim 1 of this proposal, we will use our animal model to identify critical bacterial determinants underlying GBS colonization of the neonatal gastrointestinal tract and the progression from asymptomatic carriage to invasive disease using both targeted (isogenic in-frame deletion) and unbiased (transposon insertional mutant library, Tn-seq) approaches. In addition to specific bacterial factors, we hypothesize that the composition and diversity of the developing intestinal microbiota influences the likelihood of sustained GBS colonization in exposed infants. We will use next generation 16S rRNA sequencing to examine impact of general (alpha and beta diversity) and species-level (candidate interbacterial interactions) characteristics of the murine intestinal microbiome on the establishment of GBS carriage. In Aim 2, we will determine the contribution of antibody-mediated protection against GBS colonization and late-onset disease. Using our animal model, we will determine the impact of active immunization strategies in vivo. At the conclusion of these aims, we will have defined the specific contributions of the host, pathogen and intestinal bacterial community to the pathogenesis of late-onset GBS infection. These data will provide much needed insight into the mechanisms of resistance to GBS colonization in exposed newborns and identify potential strategies to disrupt colonization in those infants most at risk for invasive disease.